Antibody screening methods

ABSTRACT

Provided are methods and compositions for the production of novel antibodies that bind specifically to a target antigen. These methods and compositions are particularly useful for producing antibodies having the antigen binding specificity of a reference antibody but with improved properties (e.g., binding affinity, immunogenicity, and thermodynamic stability) relative to the reference antibody.

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.15/847,203, filed Dec. 19, 2017, which is a division of U.S. patentapplication Ser. No. 14/005,085, filed Dec. 24, 2013, now U.S. Pat. No.9,880,160, which is the U.S. national stage of International PatentApplication Serial No. PCT/US2012/029086, filed Mar. 14, 2012, whichclaims priority to U.S. Provisional Patent Application Serial Nos.61/566,778, filed Dec. 5, 2011; and 61/453,106, filed Mar. 15, 2011, theentire disclosures of which are hereby incorporated herein by reference.

BACKGROUND

Monoclonal antibodies are hugely important as research tools,diagnostics and therapeutics. This is, in large part, due to the factthat monoclonal antibodies can be selected to bind with high specificityand affinity to almost any structural epitope.

Classical methods of immunizing animals to obtain antibodies are slowand cumbersome and, as a result, many in vitro selection techniques havebeen now developed. Examples of the techniques include nucleic aciddisplay, phage display, retroviral display, and cell surface display(e.g., yeast, mammalian, and bacterial cells). In spite of thesetechnological developments, it is still relatively difficult to obtainantibodies that possess the desired kinetic properties, selectivity,biophysical properties, and immunogenicity necessary for therapeuticuse.

Accordingly, there is a need in the art for improved methods for theselection of antibodies against a desired target.

SUMMARY OF THE INVENTION

The invention provides methods and compositions for the production ofnovel antibodies that bind specifically to a target antigen. Theinvention is particularly useful for producing novel antibodies havingthe antigen binding specificity of a reference antibody but withimproved properties (e.g., binding affinity, immunogenicity, andthermodynamic stability) relative to the reference antibody. Inparticular, the methods disclosed herein make it possible to rapidlygenerate an entirely novel antibody molecule starting only from a CDR3of known antigen-binding specificity.

The disclosed methods also allow for the rapid identification of novelpairs of VH and VL domains having a high intrinsic thermostability.Prior art methods for selecting VH/VL binding pairs generally involvethe enforced covalent linkage of VH and VL domains (e.g., as Fab or scFvfragments) and selection of VH/VL pairs based solely upon bindingaffinity of the linked VH and VL domains to a target antigen, withoutany being paid attention to the strength of non-covalent interactionbetween VH and VL domains. In contrast, the methods disclosed hereinselect stable VH/VL pairs based upon the strength of the non-covalentinteraction between unpaired VH and VL domains (in addition to thebinding affinity for a target antigen). As a result, the novel methodsdisclosed herein yield VH/VL pairs with greater intrinsicthermostability than those obtained using prior art methods.

Accordingly, in one aspect the invention provides a method for producinga V domain that binds specifically to a target antigen. The methodgenerally comprises: (a) providing a library of chimeric, unpaired VH orVL domains wherein diversity lies in the FR1-FR3 regions of each domain,and wherein each member of the library comprises the CDR3 regionsequence from a reference antibody that binds specifically to theantigen; (b) contacting the library with the antigen; and (c) selectingfrom the library at least one chimeric, unpaired VH or VL domain thatbinds specifically to the antigen, thereby producing a V domain thatbinds specifically to the antigen.

In certain embodiments, the method further comprises introducingadditional amino acid sequence diversity into the library of step (a).In one embodiment, additional amino acid sequence diversity isintroduced by random mutagenesis.

In certain embodiments, the method further comprises the step of (d)introducing additional amino acid sequence diversity into the VH or VLdomain(s) selected in step (c).

In certain embodiments, the CDR3 region sequence is from a rodent,lagomorph, avian, camelid, shark, or human antibody.

In certain embodiments, each member of the library comprises anidentical CDR3 region sequence.

In certain embodiments, the FR4 region sequences of said domains arehuman sequences.

In certain embodiments, the FR1-FR3 region sequences of the VH and VLdomains are human sequences.

In certain embodiments, each member of the library comprises FR1-FR3sequences encoded by a single human antibody VH or VL gene.

In certain embodiments, the library is a nucleic acid display librarye.g., a dsDNA display library.

In another aspect, the invention provides library of chimeric, unpairedVH or VL domains wherein diversity lies in the FR1-FR3 regions of saiddomains, and wherein each member of the library comprises the CDR3region sequence from the VH or VL domain of a reference antibody.

In certain embodiments, the CDR3 region sequence is from a rodent,lagomorph, avian, camelid, shark, or human antibody.

In certain embodiments, each member of the library comprises anidentical CDR3 region sequence.

In certain embodiments, the FR4 region sequences of said domains arehuman sequences.

In certain embodiments, the FR1-FR3 region sequences of the VH and VLdomains are human sequences.

In certain embodiments, each member of the library comprises FR1-FR3sequences encoded by a single human antibody VH or VL gene.

In certain embodiments, the library is a nucleic acid display librarye.g., a dsDNA display library.

In another aspect, the invention provides a method for selecting astable VH/VL pair. The method generally comprises: (a) providing a VHdomain that binds specifically to an antigen; (b) contacting the VHdomain with a library of VL domains such that a library of VH/VL pairsis formed; (c) contacting the library of VH/VL pairs with the antigen;and (d) selecting from the library of VH/VL pairs at least one VH/VLpair that binds specifically to the antigen, thereby selecting a stableVH/VL pair.

In certain embodiments, the method further comprises the step ofintroducing additional amino acid sequence diversity into library of VLdomains of step (b). In one embodiment, the additional amino acidsequence diversity is introduced by random mutagenesis.

In certain embodiments, the library of VL domains of step (b) compriseshuman VL domains.

In certain embodiments, the library of VL domains or VH/VL pairs is anucleic acid display library, e.g., a dsDNA display library.

In certain embodiments, the complementary VH domain of step (a) isproduced by the methods disclosed herein.

In another aspect, the invention provides a method for selecting abispecific, stable VH/VL pair. The method generally comprises: (a)providing a VH domain that binds specifically to a first antigen; (b)contacting the VH domain with a library of VL domains such that alibrary of VH/VL pairs is formed; (c) contacting the library of VH/VLpairs with a second antigen; (d) selecting from the library of VH/VLpairs at least one VH/VL pair that binds specifically to the secondantigen; and (e) contacting the VH/VL pair(s) selected in step (d) withthe first antigen; and (f) selecting at least one VH/VL pair that bindsspecifically to the first antigen, thereby selecting a bispecific,stable VH/VL pair.

In certain embodiments, the method further comprises the step ofintroducing additional amino acid sequence diversity into library of VLdomains of step (b). In one embodiment, the additional amino acidsequence diversity is introduced by random mutagenesis.

In certain embodiments, the library of VL domains of step (b) compriseshuman VL domains.

In certain embodiments, the library of VL domains or VH/VL pairs is anucleic acid display library, e.g., a dsDNA display library.

In certain embodiments, the complementary VH domain of step (a) isproduced by the methods disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of exemplary CDR3/frameworkshuffling methods as disclosed herein.

FIG. 2 is a schematic representation of the construction of exemplary VHnucleic acid display libraries for use in the disclosed methods.

FIG. 3 is a schematic representation of exemplary nucleic acid displaymethods for use in the disclosed methods. The letter “b” representsbiotin and the circles represent a tetrameric biotin binding molecule.

FIG. 4 depicts the results of in vitro binding assays measuring thebinding to human or mouse PDGFRβ of the XB1511VH domain, an unselectedXB1511 CDR3/framework shuffled DNA display library (R0), and an XB1511CDR3/framework shuffled DNA display library pool after four rounds ofselection (R4).

FIG. 5 depicts the results of surface Plasmon resonance binding studiesmeasuring the binding kinetics of XB1511 and the framework shuffledderivatives XB2202 and XB2708 to human PDGFRβ.

FIGS. 6A-6B depict the results of surface plasmon resonance bindingassays measuring the binding kinetics of XB2202 to human (FIG. 6A) andmouse (FIG. 6B) PDGFRβ.

FIGS. 7A-7B depict the results of surface plasmon resonance bindingassays measuring the binding kinetics of XB2708 to human (FIG. 7A) andmouse (FIG. 7B) PDGFRβ.

FIG. 8 is a schematic representation of the construction of exemplary VLnucleic acid display libraries for use in the disclosed methods.

FIG. 9 is a schematic representation of exemplary methods foridentification of stable VH/VL pairs.

FIG. 10 depicts the results of ELISA assays measuring the binding tohuman PDGFRβ of XB1511/VL scFv comprising VL isolated from the secondround screening pool of a VH/VL pairing DNA display screen.

FIG. 11 depicts the results of ELISA assays measuring the binding tohuman PDGFRβ of XB1511/VL scFv comprising VL isolated from the thirdround screening pool of a VH/VL pairing DNA display screen.

FIG. 12 depicts the results of ELISA assays measuring the binding tohuman PDGFRβ of XB2202/VL scFv comprising VL isolated from the secondround screening pool of a VH/VL pairing DNA display screen.

FIG. 13 depicts the results of ELISA assays measuring the binding tohuman PDGFRβ of unpaired VL from the XB1511/VL scFv set forth in FIG. 9.

FIG. 14 depicts the results of solution binding affinity studiesmeasuring the binding to human PDGFRβ of ³⁵S Met labeled XB1511 VHdomain and XB1511-containing scFV obtained from VH/VL pairing DNAdisplay screens.

FIG. 15 depicts the results of solution binding affinity studiesmeasuring the binding to human PDGFRβ of ³⁵S Met labeled XB2202 VHdomain and XB2202-containing scFV obtained from VH/VL pairing DNAdisplay screens.

FIG. 16 depicts the results of label-free migration assays measuring theability of an XB1511-containing IgG1 to inhibit the migration of humanforeskin fibroblasts.

FIG. 17 depicts the results of ELISA assays measuring the binding tohuman PDGFRβ of XB2202 VH and XB2202/A4 scFv after incubation at varioustemperatures.

DETAILED DESCRIPTION

The invention provides methods and compositions for the production ofnovel antibodies that bind specifically to a target antigen. Theinvention is particularly useful for producing antibodies having theantigen binding specificity of a reference antibody but with improvedproperties (e.g., binding affinity, immunogenicity, and thermodynamicstability) relative to the reference antibody.

I. DEFINITIONS

As used herein, the terms “VH domain” and “VL domain” refer to singleantibody variable heavy and light domains, respectively, comprising FR(Framework Regions) 1, 2, 3 and 4 and CDR (Complementary DeterminantRegions) 1, 2 and 3 (see e.g., Kabat et al. (1991) Sequences of Proteinsof Immunological Interest. (NIH Publication No. 91-3242, Bethesda). TheCDR boundaries can be defined using any art recognized numbering system.

As used herein, the term “V domain” refers to a single polypeptidecomprising a VH domain or VL domain that is devoid of constant regionsequences that facilitate the covalent pairing of said VH domain or VLdomain with a complementary VL domain or VH domain, respectively.

As used herein, the term “FR1-FR3” refers to the region of a VHencompassing FR1, CDR2, FR2, CDR2 and CDR3, but excluding the CDR3 andFR4 regions.

As used herein, the term “chimeric” refers to an antibody variabledomain comprising amino acid sequences from two or more differentantibody variable domain, e.g., a variable domain with CDR3 sequencesfrom a reference antibody and FR1-FR3 sequences from one or moredifferent antibodies.

As used herein, the term “unpaired” refers to VH or VL that are notlinked (either covalently or non-covalently) to a complementary VL or VHdomain, respectively.

As used herein, the term “complementary VL or VH domain” refers to a VLor VH domain that associates with a VH or VL domain, respectively, toform a VH/VL pair.

As used herein, the term “VH/VL pair” refers to a non-covalent dimer ofa single VH and a single VL domain, wherein the VL and VH domains areassociated through the natural VH/VL dimer interface in a similar mannerto that observed in a complete, tetrameric immunogobulin molecule, andthe dimer can bind specifically to at least one target antigen. A“stable VH/VL pair” is a VH/VL pair that does not exhibit significantdissociation of the substituent VH and VL domains under physiologicalconditions.

As used herein, the term “bispecific” refers to a VH/VL pair, whereinthe VH and the VL domains bind to different antigens.

As used herein, the term “nucleic acid display library” refers to anyart recognized in vitro cell-free phenotype-genotype linked display,including, without limitation those set forth in, for example, U.S. Pat.Nos. 7,195,880; 6,951,725; 7,078,197; 7,022,479; 6,518,018; 7,125,669;6,846,655; 6,281,344; 6,207,446; 6,214,553; 6,258,558; 6,261,804;6,429,300; 6,489,116; 6,436,665; 6,537,749; 6,602,685; 6,623,926;6,416,950; 6,660,473; 6,312,927; 5,922,545; and 6,348,315, and inWO2010/011944, which are all hereby incorporated by reference in theirentirety.

As used herein, the term “specifically binds to” refers to the abilityof a binding molecule (e.g., a VH or VL domain) to bind to an antigenwith an affinity of at least about 1×10⁻⁶ M, 1×10⁻⁷ M, 1×10⁻⁸ M, 1×10⁻⁹M, 1×10⁻¹° M, 1×10⁻¹¹M, 1×10⁻¹² M, or more, and/or bind to a target withan affinity that is at least two-fold greater than its affinity for anonspecific antigen.

As used herein, the term “antigen” refers to the binding site or epitoperecognized by a binding molecule (e.g., a VH or VL domain).

As used herein, the term “reference antibody” refers to any antibodythat binds specifically to an antigen of interest.

As used herein, the term “antibody” refers to IgG, IgM, IgA, IgD or IgEor an antigen-binding fragment thereof (e.g. VH and/or VL), whetherderived from any species naturally producing an antibody, or created byrecombinant DNA technology.

II. METHODS FOR CDR3/FRAMEWORK SHUFFLING OF ANTIBODY VARIABLE DOMAINS

In one aspect, the invention provides a method for producing a V domainthat binds specifically to a target antigen. This method allows for therapid generation of an entirely novel antibody molecule starting onlyfrom a CDR3 of known antigen-binding specificity. The method generallycomprises the steps of: (a) providing a library of chimeric, unpaired VHor VL domains wherein diversity lies in the FR1-FR3 regions of eachdomain, and wherein each member of the library comprises a CDR3 regionsequence that binds specifically to the antigen; (b) contacting thelibrary with the antigen; and (c) selecting from the library at leastone chimeric, unpaired VH or VL domain that binds specifically to theantigen.

The CDR3 region sequence can be artificial, naturally derived, or acombination thereof. Naturally derived CDR3 region sequences can be fromany organism that produces an antibody including, but not limited to,rodent, lagomorph, avian, camelid, shark, or human.

The CDR3 sequence can be from an antibody heavy chain or a light chain.However, the skilled worker will appreciate that the CDR3 must match theV-domain context into which it is placed e.g., a heavy chain CDR3 shouldbe used in a VH domain library and a light chain CDR3 should be used ina VL domain library.

In certain embodiments, the library contains a single species of CDR3sequence. In a particular embodiment, the CDR3 sequence is from a singlereference antibody that binds to the target antigen. In otherembodiments, the library contains multiple species of CDR3 sequence. Ina particular embodiment, the multiple species of CDR3 sequence arevariants of a single CDR3 sequence from a single reference antibody.Such variants can be produced by alteration (e.g. substitution,addition, deletion and/or modification) of at least one amino acid inthe CDR3 sequence from the reference antibody. Alterations can begenerated randomly (e.g., by random mutagenesis) or in a site-directedfashion using any art recognized means.

In general, the library comprises a diversity plurality of diverseFR1-FR3 region amino acid sequences. The plurality of FR1-FR3 regionamino acid sequences can be from any source including, withoutlimitation, naturally-occurring variable regions (e.g, from the antibodyVH or VL gene repertoire of an animal), artificial antibody variableregions, or a combination thereof. In certain embodiments, each memberof the library comprises FR1-FR3 sequences encoded by a single antibodyVH or VL gene (e.g., a human VH or VL gene). In other embodiments, theFR1-FR3 region sequences are human sequences. In a particularembodiment, the FR1-FR3 region sequences are naturally-occurring humanantibody variable region sequences from the B-cells of healthy donors.

In certain embodiments, the V domain further comprises an FR4 region.The FR4 region can be from the reference antibody or from anothersource. Suitable sources include, without limitation, naturallyoccurring human antibody variable regions, artificial antibody variableregions, or a combination thereof.

The V-domain library can be generated using any art recognized methodsincluding, without limitation, ab initio nucleic acid synthesis and DNAor RNA polymerase-mediated assembly. In a preferred embodiment, thelibrary is assembled by PCR using overlapping oligonucleotides. Suitableoligos for assembling a VH domain library include those set forth in SEQID No: 4-21. Suitable oligos for assembling a VL domain library includethose set forth in SEQ ID No: 70-102.

The chimeric, unpaired VH or VL domain(s), selected using the methods ofinvention, can be paired with a complementary VL or VH, respectively, togenerate a VH/VL pair using the methods disclosed herein.

III. METHODS FOR IDENTIFICATION OF STABLE VH/VL PAIRS

In another aspect, the invention provides a method for selecting stableVH/VL pairs. This method selects stable VH/VL binding pairs based uponthe strength of the non-covalent interaction between unpaired VH and VLdomains and allows for the rapid identification of novel pairs of VH andVL domains having a high intrinsic thermostability. The method generallycomprises the steps of: (a) providing a VH domain that bindsspecifically to an antigen; (b) contacting the VH domain with a libraryof VL domains such that a library of VH/VL pairs is formed; (c)contacting the library of VH/VL pairs with the antigen; and (d)selecting from the library of VH/VL pairs at least one VH/VL pair thatbinds specifically to the antigen.

The VH domain that is used to screen for stable VH/VL pairs can be fromany source (artificial, naturally derived, or a combination thereof). Incertain embodiments, the VH domain is a from a reference antibody. Inother embodiments, the VH domain is a chimeric VH domain selected usingthe CDR3/Framework methods disclosed herein.

The library of VL domains that is used to screen for stable VH/VL pairscan be from any source (artificial, naturally derived, or a combinationthereof). In certain embodiments, the library of VL domains is a humanVL domain library. In a particular embodiment, the human VL domainlibrary comprises naturally-occurring human antibody VL region sequencesfrom the B-cells of healthy donors. The VL domain libraries disclosedherein are particularly suitable for use in these methods.

Although it is preferred to screen a library of VL domains using a VHdomain as the bait, the skilled worker will appreciate that it isentirely within the scope of the invention to perform the conversescreen i.e., to screen a library of VH domains using a VL domain as thebait. This method general comprises that steps of: (a) providing a VLdomain that binds specifically to an antigen; (b) contacting the VLdomain with a library of VH domains such that a library of VH/VL pairsis formed; (c) contacting the library of VH/VL pairs with the antigen;and (d) selecting from the library of VH/VL pairs at least one VH/VLpair that binds specifically to the antigen, thereby selecting a stableVH/VL pair.

In another aspect, the invention provides, a method for selecting astable VH/VL pair, wherein the method generally comprises: (a) providinga first nucleic acid display library of VH domains, wherein members ofthe library comprise a VH domain linked to its cognate nucleic acidcoding sequence; (b) providing a second nucleic acid display library ofVL domains, wherein members of the library comprise a VL domain linkedto its cognate nucleic acid coding sequence; (c) contacting the firstnucleic acid display library with the second nucleic acid displaylibrary such that a library of VH/VL pairs is formed; (d) contacting thelibrary of VH/VL pairs with an antigen; and (e) selecting from thelibrary of VH/VL pairs at least one VH/VL pair that binds specificallyto the antigen, and, (f) ligating together the nucleic acid codingsequences of the VH and VL domains in the VH/VL pairs selected in step(e). This method is particularly advantageous in that it allows for thesimultaneous screening of VH and VL domain libraries and the precisedetermination of the sequences of the VH and VL domains in eachselected, stable VH/VL pair.

The nucleic acid coding sequences in each selected VH/VL pair can beligated together using any appropriate art recognized means (e.g.,chemical and/or enzymatic). RNA can be ligated with, for example, RNAligase. DNA can be ligated, with, for example, DNA ligase. Ligation ofthe VH and VL domain nucleic acid coding sequences can be facilitated bythe use of, for example, nucleic acid linkers, adaptors and/orrestriction enzyme digestion. In certain embodiments, the nucleic acidcoding sequences of the VH and VL domain in each selected VH/VL pair areligated together to form a single, continuous nucleic acid sequence(linear or circular), and the VH and VL domain sequences present in thecontinuous nucleic acid sequence are determined. Nucleic acid sequencedetermination can achieved by any art-recognized means, includingwithout limitation, single molecule DNA sequencing.

The above methods for identification of stable VH/VL pairs areparticularly suitable for use in combination with the CDR3/frameworkshuffling methods disclosed herein. This combination of methods allowsselection of a novel, stable VH/VL pair specific for a target antigenusing only an antigen-specific CDR3 sequence as a starting point.

The methods of the invention are particularly useful for screening forstable VH/VL pairs. However, one skilled in the art will appreciate thatthe invention can be used more broadly to screen for any stable proteindimer, wherein at least one monomer of the dimer binds to a ligand.Suitable protein dimers include, without limitation, immunoglobulinsuperfamily members (e.g., T-cell receptors), hormones, cytokines,transcription factors, and the like.

IV. METHODS OF PRODUCING BISPECIFIC ANTIBODIES

In another aspect, the invention provides a method for selecting abispecific, stable VH/VL pair, (e.g., a VH/VL pair in which the VH andVL domains bind to two different antigens). The method generallycomprises the steps of: (a) providing a VH domain that bindsspecifically to a first antigen; (b) contacting the VH domain with alibrary of VL domains such that a library of VH/VL pairs is formed; (c)contacting the library of VH/VL pairs with a second antigen; (d)selecting from the library of VH/VL pairs at least one VH/VL pair thatbinds specifically to the second antigen; and (e) contacting the VH/VLpair(s) selected in step (d) with the first antigen; and (f) selectingat least one VH/VL pair that binds specifically to the first antigen.

In one embodiment, the first and second antigens are different epitopespresent in a single molecule. In another embodiment, the first andsecond antigens are epitopes present in two separate molecules.

In certain embodiments, it is desirable to select for a promiscuouslight chain that can bind to at least two different VH domains (withdifferent antigen specificities) but that cannot, itself, specificallybind to an antigen (e.g., has a low affinity for all antigens). Suchlight chains are particularly useful in that they can be used in theassembly of full-length, bispecific, heterotetrameric antibodies. Thesebispecific antibodies generally comprising a first heavy chain with afirst antigen specificity, a second heavy chain with second antigenspecificity, and two molecules of the selected promiscuous light,wherein each heavy chain is paired with a different promiscuous lightchain molecule.

V. LIBRARY SCREENING METHODS

The skilled worker will appreciate that any type of VH or VL domainexpression library can be employed in the methods of the invention.Suitable expression libraries include, without limitation, nucleic aciddisplay, phage display, retroviral display, and cell surface displaylibraries (e.g., yeast, mammalian, and bacterial cells). In certainembodiments, the library is a nucleic acid display library. In apreferred embodiment, the nucleic acid display library is a DNA-displaylibrary constructed as exemplified herein or in WO2010/011944, which ishereby incorporated by reference in its entirety.

Methods for screening expression libraries are well known in the art.See, for example, Antibody Engineering: Methods and Protocols. Methodsin Molecular Biology Volume 248, (B.K.C. Lo, Ed) Humana Press, 2004(ISBN: 1-58829-092-1), which is hereby incorporated by reference in itsentirety. Suitable methods of screening nucleic acid display libraries,include, without limitation those set forth in U.S. Pat. Nos. 7,195,880;6,951,725; 7,078,197; 7,022,479; 6,518,018; 7,125,669; 6,846,655;6,281,344; 6,207,446; 6,214,553; 6,258,558; 6,261,804; 6,429,300;6,489,116; 6,436,665; 6,537,749; 6,602,685; 6,623,926; 6,416,950;6,660,473; 6,312,927; 5,922,545; and 6,348,315, and in WO2010/011944,which are all hereby incorporated by reference in their entirety. In apreferred embodiment, the screening of nucleic acid-display libraries isperformed as exemplified herein or in WO2010/011944.

The methods of the invention are particularly well suited to generatingVH or VL domains, and/or VH/VL pairs that bind to a target antigen withhigh affinity. To enhance affinity for the antigen, multiple rounds oflibrary screening can be performed, with additional amino acid sequencediversity being introduced at each screening round, if desired. Ifdesired, the stringency of the method can be enhanced by altering theassay conditions to reduce the affinity of the VH or VL domains for theantigen, for example, by altering the pH and/or salt concentration ofthe antigen binding reaction. Such methods selectively enrich for VH orVL domains with the highest affinity and stability.

The methods of the invention also allow for selection of VH or VLdomains, and/or VH/VL pairs having a different antigen specificity tothat of a starting reference antibody. For example, the HCDR3 from anantibody that only binds to human PDGFRβ can be used to select for a VHdomain, and/or VH/VL pair that binds to both human and mouse PDGFRβ.Such a selection is exemplified in Example 2 herein.

In certain embodiments, additional amino acid sequence diversity isintroduced into the VH or VL domain library, or the VH or VL domain(s)selected from the library. Amino acid sequence diversity can beintroduced by any art-recognized means. In certain embodiments, aminoacid sequence diversity is introduced by alteration of the nucleic acidsequence(s) encoding the VH or VL domain library, or the VH or VLdomain. In certain embodiments amino acid sequence diversity isintroduced by random mutagenesis. Such random mutagenesis can beachieved, for example, by low-fidelity PCR amplification of the nucleicacid sequence(s) encoding the VH or VL domain library, or the VH or VLdomain.

VI. ANTIBODY FORMATS

The VH and/or VL domains employed in the methods of the invention can beused in isolation or fused to additional amino acids (e.g., epitopetags) and/or domains. In certain embodiments, at least one VH domain ina library is fused to at least one CH1 domain, CH2 domain, CH3 domain ora combination thereof. In a particular embodiment, at least one VHdomain in a library is fused to at least one heavy chain constant regioncomprising at least one CH1 domain, CH2 domain and CH3 domain. Incertain embodiments, at least one VL domain in a library is fused to atleast one light chain constant region.

VH or VL domains, and/or VH/VL pairs selected using the methods of theinvention can be incorporated into another antibody format including,without limitation, scFv, Fab, and/or complete tetrameric antibody.

VII. EXAMPLES

The present invention is further illustrated by the following exampleswhich should not be construed as further limiting. The contents ofSequence Listing, figures and all references, patents and publishedpatent applications cited throughout this application are expresslyincorporated herein by reference.

Example 1. Isolation of VH Domains that Bind Specifically to HumanPDGFRβ

VH domains that bind specifically to human PDGFRβ were selected usingDNA display as set forth in WO2010/011944, which is hereby incorporatedby reference in its entirety. Specifically, a naïve, human VH domain DNAdisplay library containing derived from 10 bone marrow donors wassubject to six rounds of selection against human PDGFRβ. The selectedbinders were cloned and sequenced. From this screen VH domain clones A4,B4 and G2 were selected, the amino acid sequences of which are set forthin Table 1.

TABLE 1 Amino acid sequences of exemplary PDGFRβ- binding VH domains.Clone SEQ ID name VH Amino Acid Sequence NO. A4QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAI 1 XB1511SWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRV TITADESTSTAYMELSSLRSEDTAVYYCAIHGGDRSYWGQGTLVTVSS B4 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAI 2SWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRV TITADESTSTAYMELSSLRSEDTAVYYCAIHGGDRSYWGQGTLVTVSS G2 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYAI 3SWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRV TITADESTSTAYMELSSLRSKDTAVYYCAIHGGDRSYWGQGTLVTVSS

Example 2. HCDR3 Shuffling

A. VH Library Construction

To screen for VH domains with improved binding characteristics, theHCDR3 sequence of clone A4 (designated XB1511) was shuffled into a naïvehuman VH library, which was further selected for binding to human andmouse PDGFRβ. Specifically, the DNA sequence coding for the HCDR3 ofclone A4 (SEQ ID NO: 1) was synthesized and assembled into a librarycomprising framework regions 1-3 of naïve human VH domains amplifiedfrom bone marrow B cells and PBMCs using framework specificoligonucleotides. Human VH framework regions 1-3 were amplified using 5′VH family-specific and 3′ generic FR3 reverse primers to create separatelibraries of VH family framework regions. The VH family frameworklibraries and the XB1511 HCDR3 were shuffled by further PCRamplification using 5′ T7TMV and 3′ XB1511 FR3CDR3FR4 oligos. This alsoadded a T7TMV promoter sequence at the 5′ end for in vitrotranscription/translation. A C-terminal Cμ3 sequence and a FLAG tag (forpurification after translation) were also added by PCR using FR4 Cu3Reverse and Y109 primers, respectively, together with the 5′ T7TMVprimer. The nucleic acid sequences of the oligonucleotides used forpreparation of the HCDR3-shuffled VH library are set forth in Table 2. Aschematic representation of the general concept of CDR3/frameworkshuffling is set forth is FIG. 1 , and a schematic representation of theVH library construction is set forth in FIG. 2 .

TABLE 2 Oligonucleotides for constructing HCDR3 shuffled VH librariesSEQ ID Oligo Sequence NO. FR3 Reverse CGCACAGTAATACACGGC 4. VH1aCAATTACTATTTACAATTACAATGCAGGTKCAGCTGGTGCAGTCTG 5. VH1bCAATTACTATTTACAATTACAATGCAGGTCCAGCTTGTGCAGTCTG 6. VH1cCAATTACTATTTACAATTACAATGSAGGTCCAGCTGGTACAGTCTG 7. VH1dCAATTACTATTTACAATTACAATGCARATGCAGCTGGTGCAGTCTG 8. VH2CAATTACTATTTACAATTACAATGCAGRTCACCTTGAAGGAGTCTG 9. VH3aCAATTACTATTTACAATTACAATGGARGTGCAGCTGGTGGAGTCTG 10. VH3bCAATTACTATTTACAATTACAATGCAGGTGCAGCTGGTGGAGTCTG 11. VH3cCAATTACTATTTACAATTACAATGGAGGTGCAGCTGTTGGAGTCTG 12. VH4aCAATTACTATTTACAATTACAATGCAGSTGCAGCTGCAGGAG 13. VH4bCAATTACTATTTACAATTACAATGCAGGTGCAGCTACAGCAGTGG 14. VH5CAATTACTATTTACAATTACAATGGARGTGCAGCTGGTGCAGTCTG 15. VH6CAATTACTATTTACAATTACAATGCAGGTACAGCTGCAGCAGTCAG 16. VH7CAATTACTATTTACAATTACAATGCAGGTGCAGCTGGTGCAATCTG 17. T7TMVUTRTAATACGACTCACTATAGGGACAATTACTATTTACAATTACA 18. XB1511TGAGGAGACGGTGACCAGGGTTCCCTGGCCCCAGTAGCTCCTGTCG 19. FR3CDR3FR4CCCCCATGTKTCGCACAGTAATACACGGC Reverse FR4 Cu3GGAGACGAGGGGGAAAAGGGTTGAGGAGACGGTGACCAG 20. Reverse Y109TTTTTTTTTTTTTTTTTTTTAAATAGCGGATGCTAAGGACGACTTG 21.TCGTCGTCGTCCTTGTAGTCGGAGACGAGGGGGAAAAGGGTB. Library Screening

The HCDR3 shuffled VH domain library was then transcribed into an mRNAlibrary and subjected to selection with dsDNA display technology as setforth in WO2010/011944. A schematic representation of the screeningmethod is set forth in FIG. 3 . The selection was carried out with humanand mouse PDGFRβ at alternate round for 4 rounds. Kinetic controlled onand off rate selection was applied at successive rounds to increase thestringency of the selection to improve the affinity. Specifically,selection was performed as follows: Round 1 (R1) with 10 nM ofimmobilized human PDGFRβ; R2 with immobilized 100 nM mouse PDGFRβ; R3with 10 nM soluble human PDGFRβ and competed with 200 nM immobilizedhuman PDGFRβ for 24 hours and 120 hours; and R4 with 10 nM mouse PDGFRβ.The R4 binding pool was subcloned for DNA sequencing. Analysis of thesequences of the R4 binding pool showed that the HCDR3 of XB1511 waspresent in a variety of different framework contexts. No wild typeparental sequence was obtained from the set of sequences analyzed. Theamino acid sequences of the selected VH domains are set forth in Table3.

TABLE 3 Amino acid sequences of exemplary PDGFRβ-binding VHdomains selected from HCDR3 shuffled VH libraries. Clone SEQ ID nameVH Amino Acid Sequence NO. XB1511 framework shuffled and selected with2 rounds on human and 2 rounds on mouse PDGFRβ targets XB2202QVQLVQSGAEVKKPGSSVRVSCKASGGTFSRHAISWVRQAPGQG 22.LEWIGGILPILKTPNYAQRFQGRVTINADESTSTVYMEMSSLRSEDTAVYYCATHGGDRSYWGQGTLVTVSS C4.QMQLVQSGAEVKKPGSSVRVSCKAGGTFSRHAISWVRQAPGQG 23.LEWIGGILPILKTPNYAQRFQGRVTINADESTSTVYMEMSGLRSEDTAVYYCATHGGDRSYWGQGTLVTVSS B12.QMQLVQSGAEVKKPGSSVRVSCKASGGTFSRHAISWVRQAPGQG 24.LEWIGGILPILKTPNYAQRFQGRVTINADESTSTVYMEMSSLRSENTAVYYCATHGGDRSYWGQGTLVTVSS D07.QMQLVQSGAEVKKPGSSVRVSCKASGGTFSRHAISWVRQAPGQG 25.LEWIGGILPILKTPNYAQRFQGRVTINADESTSTVYMEMSSLRSDDTAVYYCATHGGDRSYWGQGTLVTVSS C05.QMQLVQSGAEVKKPGSSVRVSCKASGGTFSRHAISWVRQAPGQG 26.LEWIGGVLPILKTPNYAQRFQGRVTINADESTSTVYMELSSLRSEDTAVYYCATHGGDRSYWGQGTLVTVSS E05.QVQLVQSGPKVKKPGSSVRVSCKASGGTFSRHAISWVRQAPGQG 27.LEWIGGILPILKTPNYAQRFQGRVTINADESTSTVYMEMSSLRSEDTAVYYCATHGGDRSYWGQGTLVTVSS E2.QMQLVQSGAEVKKPGASVKISCKTSGYTFTDYYIQWVRQAPGQG 28.LEWVGWINPNSGNTGYAQKFQGRVTMTRDTSISTAYMELSSLRSEDTAVYYCATHGGDYSYWGQGTLVTVSS A3.QVQLVQSGAEVKKPGASVRVSCKASGYTFSDYYIQWVRQAPGQG 29.LEWMGWINPNSGGTYFAQKFQGRVTMTRDTSISTAYMELSSLTSDDTAVYYCATHGGDRGYWGQGTLVTVSS C3.QMQLVQSGAEVKKPGASVKVSCKASGYTFTDYYIQWVRQAPGQG 30.LEWIGGILPILKTPNYAQRFQGRVTINADESTSTVYMEMSSLRSEDTAVYYCATHGGDRSYWGQGTLVTVSS F10.QMQLVQSGAEVKKPGASVKVSCKASGYTFTDYYIQWVRQAPGQG 31.LEWMGWINPDSGGTYFAQKFQGRVAMTRDTSINTAYMELSSLRSDDTAVYYCATHGGDRSYWGQGTLVTVSS C12.QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYIQWVRQAPGEG 32.LEWMGWMNPDSGGTIYAQKFQGRVTMTRDTSISTAYMELSRLRPDDTAVYYCATHGGDRSYWGQGTLVTVSS H2.QMQLVQSGAEVKNPGASVKVSCKASGYPFSAYYIQWVRQAPGQG 33.LEWMGWLNPNSGDTHSAQKFQGRVTMTRDTSISTAYMELSGLTSDDTAVYYCATHGGDRSYWGQGTLVTVSS F11.QMQLVQSGAEVKNPGASVKVSCKASGYPFSAYYIQWVRQAPGQG 34.LEWMGWLNPNSGDTHSAQKFQGRVTMTRDTSISTAYMELSGPTSDDTAVYYCATHGGDRSYWGQGTLVTVSS B1.QMQLVQSGAEVRKPGASVKVSCKASGYSFSDYYIHWVRQAPGQG 35.LEWIGWINPNNGNTTYAQKFQGRVTMIRDTSISTAYMELSELRSDDTAVYYCATHGGDRSYWGQGTLVTVSS E11.QVQLVQSGAEVEKPGASVKVSCKASGYTFTDYYIHWVRQAPGQG 36.LEWMRGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCATHGGDRSYWGQGTLVTVSS H1.EVQLLESGAEVKQPGASVKVSCKTSGYTFTDYHLHWVRQAPGQG 37.LEWMGWINPNSGGTNSAPKFQGRVTMTRDTSISTAYMELSGLTSDDTAVYYCATHGGDRSYWGQGTLVTVSS E6.QMQLVQSGAEVKRPGASVKVPCKASGYTFTDYYLHWVRQAPGQG 38.LKWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSEDTAVYYCATHGGDRSYWGQGTLVTVSS A1.QVQLVQSGPEVKKPGTSVKVSCKASGFTFTSSAVQWVRQARGQR 39.LEWIGWIVVGSGNTNYAQKFQERVTITRDMSTSTAYMELSSLRSEDTAVYYCATHGGDRSYWGQGTLVTVSS H7.QVQLVQSGPEVKKPGTSVKVSCKASGFTFTSSAMQWVRQARGQR 40.LEWIGWIVVGSGNTNYAQKFQERVTITRDMSTSTAYMELSSLRSEDTAVYYCATHGGDRSYWGQGTLVTVSS G04.QVQLVQSGAEVKKPGASVKVSCKASGFTFTSYAISWVRQARGQR 41.LEWIGWIVVGSGNTNYAQKFQERVTITRDMSTSTAYMELSSLRSEDTAVYYCATHGGDRSYWGQGTLVTVSS B2.QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYQVQWVRQAPGQG 42.LEWLGVINTGVGSTNYAQKFQGRVTMTRDTATSTVYMELSSLRSEDTAVYYCATHGGDRSYWGQGTLVTVSS A7.QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYPVQWVRQAPGQG 43.LEWLGVINTGVGSTNYAQKFQGRVTMTRDTATSIVYMELSSLRSEDTAVYYCATHGGDRSYWGQGTLVTVSS H3.QVQLVQSGAEVKKPGASVKVSCRASGYTFTNSFMQWVRQVPGQR 44.LEWVGLSNPSGDYTVYAPKFQGRVTMTTDTATSTFYMELFSLRSDDTAVYYCATHGGDRSYWGQGTLVTVSS B4.QVQLVQSGAEVKKPGASVKVSCRASGYTFTNSFMQWVRQVPGQR 45.LEWVGLSNPSGDYTVYAPKLQGRVTMTTDTATGTFYMELFSLRSDDTAVYYCATHGGDRSYWGQGTLVTVSS H05.EVQLVQSGGGVVQPGGSLRLSCAASGFTFRSYGMHWVRQAPGKG 46.LEWVAFILFDGNNKYYADSVKGRFTISSDNSKNTLYLQMNSLRAEDTAVYYCATHGGDRSYWGQGTLVTVSS D06.QVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKG 47.LEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHGGDRSYWGQGTLVTVSS F3.QVQLVQSGAEVKKPGASVKVSCKASGYTFISHGMSWVRQAPGQG 48.LEWMGWISADNGNTNYAQKFQERVTITRDMSTSTAYMELSSLRSEDTAVYYCATHGGDRSYWGQGTLVTVSS A12.QVQLVQSGAEVKKPGASVKVSCKASGYTFISHGMSWVRQAPGQG 49.LEWMGWISADNGNTKYAQKFQDRVTLTTDTSTSTAYLELRSLRSDDTAVYYCATHGGDRSYWGQGTLVTVSS G3.QVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQAPGKG 50.LEWMGGFDPEDGETIYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCATHGGDRSYWGQGTLVTVSS F05.QVQLVQSGAEVKRPGASVKVSCKASGYTLTELSMHWVRQAPGKG 51.LEWMGGFDPEDGETIYAQKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCATHGGDRSYWGQGTLVTVSS H12.QVQLVQSGAEVKKPGASVKVSCKASGYTFTDNYVHWVRQAPGQG 52.LEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCATHGGDRSYWGQGTLVTVSS G12.QVQLVQSGAEVKKPGSSVKVSCKASGGAFNAYPISWVRQAPGQG 53.LEWMGGIIPVSGTPNYAQKFQGRVTITADKSTYTAYMELSSLRSEDTAVYYCATHGGDRSYWGQGTLVTVSS C06.QMQLVQSGAEVKKPGASVKVSCMASGYTFTGHYIHWVRQAPGQG 54.LEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYTELSSLRSEDTAVYYCATHGGDRSYWGQGTLVTVSS C11.QVQLVQSGAAVKKPGASVKVSRKASGYTFTNDYIHWVRQAPGQG 55.LEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCATHGGDRSYWGQGTLVTVSS F08.QVQLVQSGAEVKKPGASVKVSCKASGYTFTSSYIHWVRQAPGQG 56.LEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAMYYCATHGGDRSYWGQGTLVTVSS E9.QVQLVESGAEVRKPGESLQISCKASGYRFTNYWIGWVRQMPGKG 57.LEWMGITYPADSTTVYSPSFQGQVTISADKSISTVFLQWSSLRSEDTAVYYCATHGGDRSYWGQGTLVTVSS E11.QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYWMHWVRQAPGKG 58.LVWVSRINNDGSSTSYADSVKGRFTISRDTAKNTLYLQMNSLRAEDTAVYYCATHGGDRSYWGQGTLVTVSS H11.QVQLLESGAEVKNPGASVKVSCKASGYPFSAYYIQWVRQAPGQG 59.LEWMGWLNPNSGDTHSAQKFQGRVTMTRDTSISTAYMELSGLTSDDTAVYYCATHGGDRSYWGQGTLVTVSS C08.EVQLLESEGGLVQPGGSLRLSCTASGFSFNAFWMHWVRQAPGKG 60.LEWVSRISIDGTTTTYADSVQGRFTISRDNARNTLYLQMNSLRAEDAAVYYCATHGGDRSYWSQGTLVTVSS XB1511 framework shuffled and selected withhuman PDGFRβ and off rate selection XB2708QVQLVQSGGGVVQPGGSLRLSCAASGFTSRSYGMHWVRQAPGKG 61.LEWVAFILFDGNNKYYADSVKGRFTISSDNSKNTLYLQMNSLRAEDTAVYYCATHGGDRSYWGQGTLVTVSS D03.QVQLVQSGGGLVQPGGSLRLSCVASGFTFGNDWMHWVRQAPGKG 62.LVWVSRINADGTSTAYAESVKGRFTVSRDNAKNTLYLQMNGLRAEDTAVYYCATHGGDRSYWGQGTLVTVSS A10.QVQLVQSGGGLVQPGRSLRLSCAASGFTFDDYAMNWVRQAPGKG 63.LEWVSLIYSDGSTYYADSVKGRFTISRDNSKKTLYLQMNNLRVE DTAVYYCATHGGDRSYWGQGTLVTVSSC09. QVQLVQSGGALVQPGGSLRLSCAASGFTLSNNAMSWVRQAPGKR 64.LEWVSAIDGSGGTTYYAGSVKGRFTISSDNSKNTVFLQMNSLPAEDTAVYYCATHGGDRSYWGQGTLVTVSS A06.QVQLVQSGGGLVQPGGSLRLSCAASGFTFSGHWMHWVRQVPGKG 65.LVWVSHISNDGSITRYADSVKGRFTVARDNAKNTMYLQMNSLRAEDTAVYYCATHGGDRSYWGQGTLVTVSS C05.QVQLVQSGGGLVKPGGSLRLSCAASGFIFSSNWMHWVRQVPGKG 66.LEWVSRIKTDGSSTSYADSVKGRFTISRDNAKNTLYLQMNSLPAEDTAVYYCATHGGDRSYWGQGTLVTVSS H01.QVQLVQSGGGLVQPGGSLRLSCAASGFTLSSDWMHWVRQAPGKG 67.LVWVSRISSDGSTTAYADSVRGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCATHGGDRSYWGQGTLVTVSS G04.QVQLVQSGGGLVQPGGSLRLSCAASGFTLSSDWMHWVRQAPGKG 68.LVWVSRISSDGSTTAYADSVRGRFTISRDNTKNTLYLQMNSLRAEDTAVYYCATHGGDRSYWGQGTLVTVSS G07.QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSDWMHWVRQAPGEG 69.LVWVSRISSDGSSTAYADSVKGRFTISRDNAKNTVSLQMNSLRAEDTAVYYCATHGGDRSYWGQGTLVTVSSC. Binding Specificity of Selected HCDR3 Shuffled VH Domains

The R4 binding pool selected above was assessed for binding to bothhuman and mouse PDGFβ using a ³⁵S Met-labelled in vitro translatedlibrary. Specifically, binding of the pool to epoxy beads, 100 nM ofhuman IgG, human PDGFβ and mouse PDGFRβ were assessed. As shown in FIG.4 , the parental XB1511 VH domain showed specific binding to humanPDGFRβ, and undetectable binding to mouse PDGFRβ. The framework shuffledpre-selected library showed weak binding to human PDGFRβ. However, incontrast, the R4 framework shuffled library showed significant bindingto both human and mouse PDGFRβ.

D. Binding Affinity of Selected HCDR3 Shuffled VH Domains

The R4 framework shuffled human and mouse PDGFβ enriched VH domain poolwas cloned into E. coli expression vectors, produced and purified. Thebinding kinetics of the VH domains to human and mouse PDFGR wasdetermined using surface Plasmon resonance on a Biacore T100. Briefly,human and mouse PDGFR-hIgG1-Fc chimeric fusion protein were separatelyimmobilized using a Series CMS sensorchip (CMS) coupled to anti-hIgG1 Fcmonoclonal antibody. For each cycle, the PDGFR fusion protein was firstcaptured, followed by the injection of VH for 115 seconds at a flow rateof 100 uL/min (association). Immediately following the association phaseis a dissociation phase of 600 seconds. The surface was regenerated ateach cycle with a single injection 3M MgCl2 (10 uL/min, 60 seconds).Multiple concentrations of VH were injected (0.55 nM-40 nM) and theresulting sensorgram were analyzed with T100 Evaluation software. Thebinding kinetics was determined using 1:1 binding curve fitting. Thebinding kinetics of VH domain clones XB2202 and XB2708 to human andmouse PDGFR are shown in FIGS. 4, 5 and 6 . These results show thatXB2202 and XB2708 have a 50-150 fold affinity improvement compared toparental XB1511. Specifically, XB2202 and XB2708 have Kds of 249 pM and93 pM, respectively and off rates (Koff) of 1.86×10′ and 9.267×10⁻⁴,respectively. Both XB2202 and XB2708 bound to human and mouse PDGFRβ. Itis of particular note that, although they shared the same HCDR3, XB2202was derived from a VH1 family germline sequence and XB2708 was derivedfrom VH3 family germline sequence.

Example 3. Identification of Stable VL/VH Pairs

A. Construction of VL DNA Libraries

Human VL libraries (Vkappa and Vlamda) were constructed from B cells ofyoung healthy donors (Allcells) by RT-PCR. To ensure the diversity ofthe library, 300 million mononuclear cells from bone marrow of eachdonor and total of 10 donors and 100 million of peripheral bloodmononuclear cells from each donor and total of 10 donors were obtainedfor naïve VH and VL library construction. A schematic of the librarygeneration method is set forth in FIG. 8 .

Oligonucleotide primers for cDNA synthesis and subsequent PCRamplification of the Vkappa and Vlamda sequences were design as setforth in Table 4. Specifically, multiple sense primers were designedfrom the Vκ and Vλ FR1 regions of each family with an upstream UTRsequence. The anti-sense primers for κ and λ gene amplification weredesigned from the constant regions nested to Cκ1 (Cκ2) or Jλ with thesame Cκ2 downstream (JλCκ2). The Vκ and Vλ libraries carry the sameC-terminal sequence for PCR amplification during the selection cycles.

mRNA was prepared with individual donors using a FastTrack mRNApreparation kit (Invitrogen) following the protocol provided by the kit.First strand cDNA was synthesized from the isolated mRNA using primersspecific for the light chain kappa and lambda constant regions (Cκ1 andCλ1).

PCR amplification of the Vkappa and Vlamda sequences was performed withCκ2 and Vκ family specific or JλCκ2 mix and Vλ family specific oligosusing cDNA synthesized from mRNA prepared from the various source of Bcells as template. The PCR was performed with individual families andindividual donors for 18-20 cycles. After gel purification, Vκ and Vλlibraries from different sources were pooled to generate Vκ and Vλlibraries.

TABLE 4 Oligonucleotides for constructing human Vλ and VκDNA display libraries SEQ ID Sequence NO. Oligo Ck1CAACTGCTCATCAGATGGCGG 70. cl1 CAGTGTGGCCTTGTTGGCTTG 71. Ck2AGATGGTGCAGCCACAGTTCG 72. Jl1-3Ck2AGATGGTGCAGCCACAGTTCGTAGACGGTSASCTTGGTCCC 73. J17Ck2AGATGGTGCAGCCACAGTTCGGAGACGGTCAGCTGGGTGCC 74. T7TMVUTRTAATACGACTCACTATAGGGACAATTACTATTTACAATTACA 75. Vλ oligos UTRVk1aCAATTACTATTTACAATTACAATGRACATCCAGATGACCCAG 76. UTRVk1bCAATTACTATTTACAATTACAATGGMCATCCAGTTGACCCAG 77. UTRVk1cCAATTACTATTTACAATTACAATGGCCATCCRGATGACCCAG 78. UTRVk1dCAATTACTATTTACAATTACAATGGTCATCTGGATGACCCAG 79. UTRVk2aCAATTACTATTTACAATTACAATGGATATTGTGATGACCCAG 80. UTRVk2bCAATTACTATTTACAATTACAATGGATRTTGTGATGACTCAG 81. UTRVk3aCAATTACTATTTACAATTACAATGGAAATTGTGTTGACRCAG 82. UTRVk3bCAATTACTATTTACAATTACAATGGAAATAGTGATGACGCAG 83. UTRVk3cCAATTACTATTTACAATTACAATGGAAATTGTAATGACACAG 84. UTRVk4aCAATTACTATTTACAATTACAATGGACATCGTGATGACCCAG 85. UTRVk5aCAATTACTATTTACAATTACAATGGAAACGACACTCACGCAG 86. UTRVk6aCAATTACTATTTACAATTACAATGGAAATTGTGCTGACTCAG 87. UTRVk6bCAATTACTATTTACAATTACAATGGATGTTGTGATGACACAG 88. UTRVL1aCAATTACTATTTACAATTACAATGCAGTCTGTGCTGACKCAG 89. UTRVL1bCAATTACTATTTACAATTACAATGCAGTCTGTGYTGACGCAG 90. UTRVL2CAATTACTATTTACAATTACAATGCAGTCTGCCCTGACTCAG 91. UTRVL3aCAATTACTATTTACAATTACAATGTCCTATGWGCTGACTCAG 92. UTRVL3bCAATTACTATTTACAATTACAATGTCCTATGAGCTGACACAG 93. UTRVL3cCAATTACTATTTACAATTACAATGTCTTCTGAGCTGACTCAG 94. UTRVL3dCAATTACTATTTACAATTACAATGTCCTATGAGCTGATGCAG 95. UTRVL4CAATTACTATTTACAATTACAATGCAGCYTGTGCTGACTCAA 96. UTRVL5CAATTACTATTTACAATTACAATGCAGSCTGTGCTGACTCAG 97. UTRVL6CAATTACTATTTACAATTACAATGAATTTTATGCTGACTCAG 98. UTRVL7CAATTACTATTTACAATTACAATGCAGRCTGTGGTGACTCAG 99. UTRVL8CAATTACTATTTACAATTACAATGCAGACTGTGGTGACCCAG 100. UTRVL4/9CAATTACTATTTACAATTACAATGCWGCCTGTGCTGACTCAG 101. UTRVL10CAATTACTATTTACAATTACAATGCAGGCAGGGCTGACTCAG 102. R = A/G, Y = C/T, K= G/T, M = A/C, S = G/C, W = A/TB. Generation of VL Fusion Libraries by dsDNA Display

Vk and VL DNA libraries generated using the methods set forth in thisExample were transcribed into mRNA libraries using the T7 Megascript kit(Invitrogen, Cat #AM1334). The mRNA was purified with RNeasy MinEluteCleanup Kit (Qiagen, Cat #74204) following protocol provided by the kit.A total of 600 pmol of RNA (300 pmol of Vk and Vl libraries) was ligatedand assembled with dsDNA display linkers and components as described inWO2010/011944. The assembled VL library was subjected to in vitrotranslation system to create a fusion library in which each VL domain(phenotype) is stably fused to its coding sequence (genotype). ³⁵S Metwas incorporated in the translation process to radiolabel the fusions.The library was then purified with oligo dT cellulose, converted into adsDNA display library using the standard molecular biology techniques ofreverse transcription, RNaseH digestion, 2^(nd) strand DNA synthesis,followed by flag tag purification. A schematic of this process is setforth in FIG. 3 .

C. Identification of VL Pairs for XB1511, and XB2202 VH Domains

As a proof of concept, PDGFRβ VH binding domain XB1511 was translated asfree protein (with incorporation of ³⁵S Met in the translation reaction)and affinity purified through a c-terminal flag tag. The XB1511 and apurified VL fusion library (prepared as above) were then mixed at anequal molar ratio and incubated at 25 C overnight to allow for in vitroassociation of VH and VL fusion domains through their hydrophobic patch.The mixture was then contacted with PDGFRβ target pre-immobilized onEpoxy450 beads or in solution and captured by protein A beads. Complexesthat bound to the immobilized PDGFRβ target were washed and eluted with0.1N KOH. PCR was performed with VL specific primer sets to recover theVLs that bound to the PDGFRβ target, both as VH-VL pairs and as unpairedVL domains. The VL pairing was performed for 3 rounds, with lowstringency (100 nM PDGFRβ) for the first 2 rounds and higher stringency(10 nM PDGFRβ) for the third round. PDGFRβ VH binding domain XB2202 wasalso paired with VL library similarly for 2 rounds. For each round ofXB2202 VL pairing and selection, the stringency was increased by kineticcontrolled on and off rate strategy to identify VLs that can pair withVH stably and enhance the VH binding. A schematic of this process is setforth in FIG. 9 .

VL pools identified above were then cloned into Blunt Zero TOPO vector(Invitrogen) and VL-encoding DNA sequences were amplified from theresultant bacterial colonies by PCR using M13 forward and reverseprimers. The individual amplified VL-encoding DNA sequences were thensequenced. The sequence data obtained from VL pools showed that adiverse repertoire of VLs was enriched through the process. Multiplefamilies and frameworks were present in the pool. Several VLs werepresent as replicates or families. Distinct VL families could beidentified and several VLs were present more than once. Exemplary VLsequences identified using the methods of the invention that pair withthe PDGFRβ-binding VH domains XB1511 and XB2202 are set forth in Table 4herein.

TABLE 4 Light chain variable domain (VL) amino acidsequences of exemplary anti-PDGFRβ antibodies. Clone SEQ ID nameVL Amino Acid Sequence NO. PR2 VL sequences from XB1511 pairing B10.QSVLTQSPDLQSVTPREKLTITCRASQTIGSTLHWYQQKPGQSPR 103LVIKYAYQSVSGVPSRFSGSGSGTEFTLTINGLEAEDAATYYCHQ SSSLPWTFGQGTKLTVL H10.QSVLTQSPDFQSVSPKDKVTITCRASQSIGSSLHWYQQKPGQSPK 104LLIKYSSQSFSGVPSRFSGSASGTEFTLTITGLEAEDAATYYCHQ SSSLPHTFGQGTKVTVL F10.QSVLTQSPEFQSVTPKEKVTITCRASQSIGSGLHWYQQKPHQSPK 105LLIRYASQSMSGVPSRFSGSGSGTDFTLTISRLEVEDAAMYYCHQ SSSLPWTFGQGTKVTVL B12.QSVLTQSPDFQSVTPKQNVTFTCRASQSIGIKLHWYQQKPDQSPK 106VLIKYASQPFSGVPSRFSGRGSGTDFTLTINSLEAEDAATYYCHQ TSSLPLTFGGGTKVTVL B11.QSVLTQSPGTLSLSPGERATLSCRASQSVSSNYLAWYQQKPGQAP 107RLLIYGASSRASGIPVRVSGSGSGTDFTLTISRLEPEDFAVYYCQ QYGSSPWTFGQGTKLTVL E7.QSVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAP 108RLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQ QYGSSPQTFGQGTKLTVL E8.QSVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAP 109RLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQ QYGSSPPYTFGQGTKLTVL H8.QSVLTQSPGTLSLSPGERATLSCRASQSVSSNYLAWYLQKPGQAP 110RLLISGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQ QYAGSPFTFGPGTKLTVL H12.QSVLTQSPGTLSLSPGERATLSCRASQSVRSSYVAWYQQKPGQAP 111RLLISGASRRATGIPDRFTGSGSGTDFTLTISRLEPEDFAVYHCQ QFGSSPWTFGQGTKLTVL F8.QSVLTQPPSASGTPGQRVTISCSGGRSNIGGNAVNWYQQKPGQAP 112RLLIHVASRRVTGIPDRFSGSGSGTDFTLTISRLEPEDFAIYYCQ QYGSSPLTFGGGTKLTVL D11.QSVLTQSPGTLSLSPGERATLSCRASQNITSNFFAWYQQKPGQAP 113RLLIYGASSRATGIPDRISGSGSGTDFTLTISRLEPEDFALYYCQ QYGASPRTFGQGTQLTVL G8.QSVLTQSPGTLSLSPGDRATLSCRASQSLSGTYLAWYQQKPGQAP 114RLLIYDASNRAAGIPKRFSGSGSRTDFTLTISRVDPADSAVYYCQ QYGSALLTFGGGTKVTVL H9.QSVLTQSPGTLSLSPGESATLSCRASEDIYNNYLAWYQHKRGQPP 115RLLIFRASTRATGIPTRFSGSGSGRDFVLTINRLEPEDFAVYYCQ QYGNSWTFGQGTKLTVL H11.QSVLTQSPDFQSVTPKEKVTITCRASQSIGSSLHWYQQKPDQSPK 116LLITFASQSFSGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCHQ SRNLPFTFGPGTKLTVL G12.QSVLTQSPDFQSVTPKEEVTITCRASESIGTALHWYQQKPDQSPK 117LLIKYSSQSISGVPSRFVGRGSETEFTLTINSLEAENAATYYCHQ SRSFPLTFGQGTQLTVL E11.QSVLTQSPGTLSLSPGERATLSCRTSQILHSQYLAWYQQKRGQAP 118RLLIFRASTRATGIPERFSGSGSGRDFVLTISRLEPEDSAVYYCQ QYETSWTFGQGTKVTVL F12.QSVLTQDPVVSVALGQTVRITCQGDTLRTCYASWYQQRPRQAPIL 119VIYGENNRPSGIPARFSGSSSGSTASLTITGAQAEDEGDYYCHCR DGLNHLVFGGGTKVTVL C8.QSVLTQPPSVSAAPGQKVTISCSGSTSNIGKNFVSWYQHLPGTAP 120KLLIYDNYQRFSGIPDRFSGFKSGTSATLSITGLQTADEADYYCG TWDSSLSVVIFGGGTKLTVL A8.QAGLTQSPDFQSVTPKERVTITCRASRYIGSNLHWYQQKPDQPPK 121LLIKLASQSFSGVPPRFSGGGSGTDFTLTINGLEAEDAATYYCHQ TGSFPYTFGQGTKLTVL B8.QAVLTQEPSLTVSPGGTVTLTCGSSTGAVTSGHSPFWFQQRPGQA 122PRTLIYDTSNKQSWTPARFSGSLLGGKAALTLSGAQPEDEAEYYC LLSYSGPRVVFGGGTKVTVL F7.QAVVTQSPDSLAVSLGERATISCKSSXSLLYRSNNKNYLAWYQQK 123PGQPPRLLISWASTRESGVPDRFSGSGSGTDFTLTVSRLRAEDAA VYYCQQSYRTPFSFGPGTKVTVLB7. SYVLTQPLSVSVALGQTARISCGGANIANKNVHWYQLQPGQAPVL 124VIYRDSNRPSGIPERFSGSNSGNTATLTITRAQARDEADYYCQVW DSSSVIIGGGTKVTVL G9.SYVLTQDPAVSVALGQTVRITCQGDSLRTYYASWYRQKPGQAPVL 125VFYGKDNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCKSR DSSAMRWVFGGGTKLTVL A9.NFMLTQEPSLTVSPGGTVTLTCGSSTGAVTSGHYPYWFQQKPGQV 126PRTFIYDTHNRHSWTPVRFSGSLFGGKAALTLSGAQPEDEAEYYC LLYFNPTRVFGGGTKLTV A11.NFMLTQPPSASASLGASVTLTCTLSSGYSNYKVDWYQQRPGKGPR 127FVMRVGTGGIVGSKGDGIPDRFSVLGSGLNRYLTIKNIQEEDESD YHCGADHGRVFGGGTKLTVL E12.QPVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYHLRPGQAPVL 128VIYFDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVW HSGVIFGGGTKLTVL H7.QPVLTQSLDFQSVTPKEKVTITCRASQNIGNSLHWYQQKPNQSPK 129VLIKYASQSFSGVPSRFSGSGFGTDFTLTINSLEPEDAATYYCHQ SRSSHTFGQGTKLTVL A10.EIVLTQSPGNLSLSPGERATLSCTRCTGNIASHFVQWYQQRPGSS 130PTTVIFGNNQRPSGVSDRFSGSIDSSSNSASLTISRLKTEDEADY YCQSFDVYSHEVVFGGGTKLTVLC11. QTVVTQTPVSLSVTPGQPASISCKSSQSLLNSDDGKTYLYWYLQR 131PGQPPHLLIYEVSKRFSGVPDRFSGSGSGTDFTLRISRVEAEDVG VFYCMQSTHFPFTFGPGTKVTVLD10. NIQMTQSPVSLSASLGDTVSITCQASHDISNYLNWYQQKPGKAPK 132LLIYDASHLEAGVPSRFRGSGSGTDFTLTINRLEPEDFAVYYCQQ YDSPPWTFGQGTKLTVL D12.DVVLTQSPGTMSLSPGERATLSCRASQSVSRTYLAWHQQKPGQAP 133RLLIYGASSRAAGIPDRFSGSGSGTDFTLSISRLEPEDFAVYYCQ QHDTSQWTFGQGTKLTVL C7.DIVMTQTPLSLSVTPGQPASISCKSSQSLLHSDGKTHLFWYLQRP 134GQSPQLLIYEVSGRFSGVSERFSGSGSGTDFTLKISRVEAEDVGV YYCMQGLHIPHTFGQGTKVEIK D7.DIVMTQSPLSLPVTLGQPASISCRSSHSLVHSDGNIYLNWYHQRP 135GQSPRRLIYSVSKRDSGVPDRFSGSGSRTDFTLKISRVEAEDVGV YFCMQSTHQWTFGQGTKVEIK C9.VIWMTQSPSTVSASVGDRVTITCRASQSISSWLAWYQQKPGKAPN 136LLIYEASRLESGIPSRFSGSGSGTEFTLTXSSLQPDDFATYYCQQ YDSYSRTFGQGTKVAIK C12.DVVMTQSPSSLSASVGDRVTITCRTSQGIRNYLSWYQQKPAKAPK 137LLIHGASGLQSGVPSRFSGSGSGTNFTLTISSLQPEDFATYYCQQ SFSMRTFGQGTKVEIK D8.EIVMTQSPGTLTLSPGEGATLSCRASQSVTSNYLAWYQQRPGASS 138LQSGQAPRLLIYDASNRATGIPDRFSGSGFGTDFTLTISRLEPED FAVYYCQQYVNSRTFGQGTKVEIKD9. EIVMTQSPVTLSVSPGERATLSCRASQSVSSKLAWYQQKPGQAPR 139LLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAIYYCQQ YNDFFTFGPGTKVDIK G7.EIVLTQTPLSSPVTLGQPASISCRSSESPVHSDGNIYLSWLHQRP 140GQPPRLLLYKISNRMSGVPDRFSGSGAGTDFTLKISRVEAEDVGV YYCMQATQFPSFGQGTKLEIK G11.EIVLTQSPGTLSLSPGEGATLSCRASQSVSSRNLAWYQQKPGQAP 141RLLIYGGSIRASGTSTRFSGSGSGTDFTLTINRLEPEDFAVYYCQ QYGDSVFTFGPGTKVDIK F9.NIQMTQSPSSLSASVGDRVNITCRASDNIGNYLNWYQHKPGKAPT 142VLIYAASTLHYGVPSRFSGRGSGTDFTVTISSLQPEDSATYYCQQ SYSTPRTFGQGTRVELK E9.AIQMTQSPSSLSASVGDRVTITCRASESISNYLNWYQQKPGKAPK 143LLLSAASRLQSGVPSRFSGSGSGTDFTLTITSLQPEDLATYYCQE SYSTLLYTFGQGTKLEIKVL sequences from XB2202 VLpairing B1.SYELTQPPSVSVAPGKTASITCGGNNIGYDSVHWYQQKPGQAPVL 144VVFDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVW ESGSEHYVFGTGTQLTVL E6.LPVLTQPPSVSVAPGQTARISCGGNNIGATTVHWYQHRPGQAPVS 145VIFYDNDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVW ESTSDHPTFGGGTQLTVL F3.QSVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVL 146VIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVW DSSSDHWVFGGGTKLTVL H4.SYELTQSPSVSVPPGQTARITCGGNNIVSKGVHWYQQRPGQAPVL 147VVYDDSDRPSGIPERFAGFNSGNTATLTISRVEAGDEADYYCQVW DSSSGHRGVFGGGTKVTVL H5.SYELTQPPSVSMAPGKTARITCGGNNLGSKIVHWYQQKPGQAPVV 148VIYSDRDRPSGVPERFSGSNSGNSATLTISGVEAGDEADYYCQVW DSATDHVVFGGGTKLTVL B5.SYELTQPPSVSVAPGQTATITCAGNNIGGKSVQWYQQKPGQAPVV 149VVYDDYGRPSGIPERVSGSNSGNTATLTLTRVEAGDEADYYCQVW DSDRHHVVFGGGTKLTVL G6.QLVLTQPPSVSVSPGQTASITCSGDNLGHTNACWYQQNPGQSPVL 150VIYQDTKRPSGIPERFSGSNSGNPATLTIXRVXAGDEANYYCQVW DINDDYAVFGTGTXLTVL C1.QSVLTQSPGTLSLSPGERATLSCTASQSVSSTYLTWYQQKPGQAP 151RLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQ QYVSSPPMYTFGQL F1.DIQMTQSPSTLSASVGDRVTISCRASQNIDYDLAWYQXKPGKAPX 152LLIYGASNLEGGVPSXFSGXGSGTEFTLTISSLQPDXSATYYCQQ YVTYPLTFGQGTRLEIK A3.AIQMTQSPSSLSASVGDRVTMTCQASQVIDKYVNWYRQRPGKAPE 153LLIYGASTLESGVPSRFSGSGSGTQFTFSITSVQPEDFATYICQQ YDSVPLTFGPGTILDVKRTVA B4.DIQLTQSPSSLSASIGDRVTITCQASQDIFHYLNWFQQKPGKAPK 154LLIYEASNLETGVPSRFSGSGSVTDFTFTISSLQPEDIATYYCQQ YEDLPSFGGGTKVDIKRTVA B6.EIVLTQSPGTLSLSPGERATLSCRASQSFGSNYLAWYQHKPGQAP 155RLLIFAASNRATGIPDRFTGSASGTDFTLTINRVEPEDLAVYYCQ QYGSFPYSFGQGTKLEIK F2.NIQMTQSPSSLSASVGDRVTITCQASQFIHIYLNWYQQKLGKAPK 156LLIYGASNLERGVPSRFSGRGSETDFTFTIDSLQPEDIATYFCQQ YQNPPFTFGGGTKVE INGTVA D3.AIRMTQSPSSLSASIGDRISVTCRASQDVGIYVAWFQQKPGKPPR 157SLIYAASYLQTAVPPKFRGSGSGTDFTLTISDLQPDDFATYYCQQ YKTFPHTFGQGTKLDFKRTVA G2.VIWMTQSPSTLSASVGDRVTITCRASQDINTWLAWYQQKPGKAPK 158LLMFKVSTLESGDFSRFSGSGSGTEFTLTVSSLQPDDSAIYYCQQ YHSYPYTFGQGTRLEIK A4.DVVMTQSPSSLSASVGDRVTITCQASQDISNWLNWYQQKPGKAPK 159LLIYEASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQ YNNVLRTFGQGTKVEIK G4.EIVMTXSPATLSVSPGERVTLSCRVSQNVFSDLAWYQRKTGQSPR 160LLIHGASTRATGIPTRFSGSGSGTEFTLTISSLXSDDFAVYYCQQ YNKWPTFGQGTKVEIK D5.AIQLTQSPSSLSASVGDRVNITCRASDNIGNYLNWYQHKPGKAPT 161VLIYAASTLHYGVPSRFSGRGSGTDFTVTISSLRSDDFAVYYCQQ YYNWPPWTFGQGTTVDIKRTVA A1.EIVLTQSPATLSLSPGERATLSCRASQSVSSFLAWYQQKPGQAPR 162LLIFEASTRATGISARFSGSGSGTDFTLTISTLEPEDFAVYYCQQ RSNGVTFGQGTRLEIK H2.DIQMTQSPSTLSASVGDTVTITCRATESISIWLAWYQQEPGKAPN 163LLVSQASSLKTGVPSRFSASGSGTEFTLTISSLHPDDFATYVCQH YHTYPFTFGPGTKVDMKRTVA E2.EIVLTQSPDSXAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQK 164PGQPPRLLIYWASTRESGVPDRFSGSGSGTDFTLTISRLQAEDVA VYYCQQYYLTPTFTVTFGQGTKLEIKF4. DIQLTQSPSSVSASVGDRVTITCRASQDISSWLAWYQQKPGKAPK 165FLIYRATNLQSGVPSRFSGSGSGTDFTLTISSLQPGDFATYYCQQ TNTFPLTFGGGTKVEVKRTVA C5.DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNRNYLAWYQKK 166PGQPPKLLFYWASTRESGVSDRFSGSGSGTDFTLTISSLQAEDVA VYYCQQYHTTPYTFGQGTKLEIKE5. VIWMTQSPSSLSASVGDRVSITCRASQTFTSHLNWYQQKPGQPPK 167LLIFAASNLQSGVPSRFSGSGSGTDFTLTINGLQATDFATYYCQQ SFSSPWTFGQGTTVDVKGTVA F6.DIQMTQSPSSLSASVGDRVTITCRASQSVNVYLNWYQQKPGKAPK 168LLIYSASTLQSGVPSRFTGSGSRTDFTLTINGLQPEDFATYYCQQ SFTTLVTFGPGTRVDVTRTVA G5.DIQMTQSPSSLSASVGDRVTITCRASQDISSSLAWYQQKPGKAPK 169PLIYDASTLQTGVPSRFSGRASGTDFTLTIDSLQPEDFATYCCQQ FNSYPLSFGGGTKVELKRTVA A5.SYELTQPPSASASLGASVTLTCTVSSGYRSYEVDWFQQRPGKGPR 170FVMRVGTGGIVGSRGDGIPDRFSVWGSGLNRYLTIEDIQEEDESD YYCGADHGSGSNLVYVFGTGTKVTVLD6. QLVLTQPPSASASLGASVTLTCTLSSDYSSYNVDWYQQRPGMGPR 171FLMRVGTGGIVGSRGDGIPDRFSVKGSGLNRYLTIKNIQEEDESD YYCGADHGSGSDFVYVFGIGTKLTVLE4. QSVLTQPPSASGTPGQRVTISCSGSSTNIGSNAVNWYQQLPRTAP 172KLLIYTNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEANYYCA AWDDSLNGPVFGGGTQLTVL F5.QSVLTQPPSASGTPGQTVIISCSGGGSNIGSNFGYWYQQFPGTAP 173KLLIYTTDRRPSGVPDRFSGSKSGTTASLAISGLRSEDEADYYCA AWDDRLSGPVFGGGTQLTVL G1.QTVVTQPPSVSGTPGQRVTISCSGSSSNIGSNSVDWYQQFPGSAP 174KLLIYTTNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCA TWDDDLSNPKWVFGGGTKLTVL E3.DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNFLDWYLQKP 175GQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGI YYCMQALQTSWTFGQGTKLEIK A2.DICRIRPLIRLTIGTITIYNYNGCCDDTVSTLPARHPWTAGLHLQ 176SPRRLMYQVSTRDSGVPDRFSGSGSGTDFTLRISRVEAEDVGVYY CMQGTHWPYTFGQGTKLEIRRTVAD1. DIVMTQTPLSLSVTPGQPAAISCKSSQSLVHRDGKTYLYWYLQKP 177GHSPQLLVYEASSRFSGVPDRISGSASGTQFTLNISRVEAEDVGL YYCMQSRNLPKTFGQGTKVEIK C4.SYELTQPTSLSASPGASASLTCTLSSGFNVVSYNIYWYQQKPGSP 178PQYLLRYRSDSDRHQGSGVPSRFSGSKDASANAGILVISALQSDD EADYYCMVWYSAWVFGGG E1.SYELTQPLSVSVALGQTATITCAGNNIGTYYVHWYQQRPGQAPVL 179VMYRDTNRPSGISDRFSGSNSGDTATLTICGVQVGDEADYYCHVL DSSTIVIFGGGTQLTVL A6.QSVLTQSPATLSVSPGERASLSCRASQSVSSNLAWYQQKPGQAPR 180LLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCHQ YNNWPLYTFGQGTKLTVL H1.QSVLTQDPAVPVALGQTVRITCQGDSLRTYYASWYQQKPGQAPLL 181VIYGKNTRPSGIPVRFSGSSSGNTASLTITGAQAEDEADYYCNSR DSSGYLLLFGTGTKLTVL B2.QAVLTQEPSLTVSPGGTVTLTCGSSTGAVTSGHYPYWFQQKPGQA 182PRTLIYDASNKHSWTPARFSGSLLGGKAALTLSGAQPEDEAEYYC LLSYSGAGVFGTGTKVTVL C2.DIQMTQSPSSLSASVGDRVAIACRPSQDIGTDLGWYQQKPGKAPK 183LLIFDSSTLQSGVPSRFSGSLSGTDFILTITNLQPEDFATYYCLQ DYSFPYTFGQGTKLQIKRTVA G3.SYVLTQPPSVSVSPGQTASITCSGDELKYKYTCWYHQKPGQSPVL 184LIYQDTKRPSGIPERFSGSRSENTATLTISGTQAMDEADYYCQAW DSSHAVFGRGTQLTVL H3.SYVLTQPPSVSVFPGQTARITCSGSTFPKLYSFWYQQKTGQAPLL 185VIYKDTERPSGIPERFSGSTSGTTVTLIISGVQPEDDADYYCQSE DSRGPVFGGGTKVTVL D4.GVVMTQTPLSSLVTLGQPASISCRSSESVVHDDGNTYLSWLQQRP 186GQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEPEDVGV YYCVQATHFPVTFGGGTRVEIK C6.QSALTQPASVSASPGQSVTISCTGTSDDVGRYDYVSWYQQHPGGA 187PKLILYDVNRRPSGVSDRFSGSKSANKASLTISGLQADDEGDYYC CSYTTGSTLYLFGTGTQLTVLD. Evaluation of Identified VH and VL Pairs

To evaluate the characteristics of the identified VH-VL pairs, 10-12scFVs from each pool were constructed and produced by either in vitrotranslation or by E. coli expression, followed by affinity purification.

A PDGFRβ binding ELISA assay was performed to assess the binding of thescFv to immobilized PDGFRβ and to determine the EC50. Specifically, 2ug/mL of human PDGFRβ and human Fc or IgG in PBS was immobilized onMaxisorp plates at 4° C. overnight. The plate was then washed andblocked with superblock. In vitro translated crude scFv lysate wasdiluted 1:3 in 1×PBST. 100 ul of the diluted scFv lysate was loaded intoeach well of Maxisorp plates and incubated for 1 hour at roomtemperature. scFv that bound to immobilized PDGFRβ was detected byanti-flag antibody-HRP at 1:5000 dilution and a TMB substrate. The platewas read on a Molecular Device plate reader with end point assay at OD450 nm. As shown in FIGS. 10, 11 and 12 , in the ELISA binding assay,greater than 50% of the scFvs generated for XB1511 and XB2202 showedspecific binding to hPDGFRβ. In contrast, the unpaired VLs alone did notshow binding to PDGFRβ (see FIG. 13 ).

The affinity of several scFvs was determined by solution basedequilibrium binding assay. Specifically, 120 pmol of scFv RNA wastranslated into free protein with ³⁵S Met incorporated. The translatedreaction mixture was 3-fold diluted in binding buffer containing 1×PBSwith 0.025% triton, 1 mg/mL BSA and 0.1 mg/mL sssDNA. Human PDGFRβ wasdiluted in the same binding buffer to final concentrations from 100 nMto 0 nM. The diluted scFv mixture was incubated with hPDGFRβ in finalvolume of 100 ul on Kingfisher plates (Thermofisher Scientific,97002084). Following incubation, 25 ul of protein A magnetic beads(Invitrogen) were used to capture the PDGFRβ from solution. The capturedPDGFRβ was washed and eluted in kingfisher Reader (ThermofisherScientific). The amount of scFv (labeled with ³⁵S Met) bound to themagnetic bead-immobilized hPDGFRβ was counted using a scintillationcounter and the Kd was calculated with Graph Pad Prism 5. For theXB1511-derived scFv tested, 2 scFv showed an 8-10 fold higher K_(D), 1showed 2.5 fold higher K_(D), and 4 showed a similar K_(D) when comparedto XB1511 VH alone (FIG. 14 ). Only 1 scFv showed a lower K_(D) thanXB1511 VH alone. As shown in FIG. 15 , both of the XB2202-derived scFvtested showed approximately an 8-10 fold better K_(D) when compared toXB2202 VH alone.

Example 4. Conversion of VH-VL Pairs to Heterotetrameric IgG andDemonstration of Biological Activity

XB1511 VH and D8 VL were expressed as heterotetrameric IgG in 293Tcells. Cell culture supernatant was collected after 48 hours and 96hours and the expressed IgG was purified with protein A agarose beads.The IgG was produced at 8 mg/L without any optimization. To evaluate thebiological activity of the XB1511/D8 IgG, HFF-1 human foreskinfibroblasts were seeded in 384-well BIND biosensors and allowed toattach overnight in serum-free media. The fibroblast cells were thenstimulated with 5 ng/mL or 10 ng/mL of PDGFBB ligand and allowed tomigrate for 18 hours. BIND Scanner images were captured every 15 minutesand software analysis tools used to measure the track lengths ofindividual cell migration responses. Track length is represented by a“heat map” from blue (no migration) to red (maximal migration). As shownin FIG. 16 , the XB1511/D8 IgG was able to completely block thePDGFBB-induced migration of human fibroblasts.

Example 5. scFv Thermostability

The thermostability of the XB2202 VH and scFv XB2202-A4 were determined.Specifically, 1 mg/mL of XB2202 and XB2202-A4 were incubated at 4 C, 37C, 60 C and 70 C for 12 hours and PDGFRβ binding ELISA was performed totest the binding activity of the protein after incubation. As shown inFIG. 17 , XB2202 VH lost significant PDGFRβ binding activity afterincubation at 60 C and completely lost binding activity after incubationat 70 C. The Tm of XB2202 was measured to be approximately 62 C. Incontrast, scFv XB2202-A4 was completely active after 12 hour incubationat 70 C, indicating that the Tm of scFv XB2202 was greater than 70 C.

We claim:
 1. A method for selecting a bispecific, stable VH/VL pair, themethod comprising: (a) providing an unpaired VH domain that bindsspecifically to a first antigen; (b) contacting the unpaired VH domainwith a library of unpaired VL domains such that a library of VH/VL pairsis formed; (c) contacting the library of VH/VL pairs with a secondantigen; (d) selecting from the library of VH/VL pairs at least oneVH/VL pair that binds specifically to the second antigen; (e) contactingthe VH/VL pair(s) selected in step (d) with the first antigen; and (f)selecting at least one VH/VL pair that binds specifically to the firstantigen, thereby selecting a bispecific, stable VH/VL pair.
 2. Themethod of claim 1, further comprising the step of introducing additionalamino acid sequence diversity into the library of unpaired VL domains ofstep (b).
 3. The method of claim 2, wherein the additional amino acidsequence diversity is introduced by random mutagenesis.
 4. The method ofclaim 1, wherein the library of unpaired VL domains of step (b)comprises human VL domains.
 5. The method of claim 1, wherein thelibrary of unpaired VL domains is a nucleic acid display library.
 6. Themethod of claim 1, wherein the unpaired VH domain of step (a) isproduced by (a) providing a library of chimeric, unpaired VH domainswherein diversity lies in the FR1-FR3 regions, and wherein each memberof the library comprises the CDR3 region sequence from the VH domain ofa reference antibody that binds specifically to the first antigen; (b)contacting the library with the first antigen; and (c) selecting fromthe library at least one chimeric, unpaired VH domain that bindsspecifically to the first antigen, thereby producing an unpaired VHdomain that binds specifically to the first antigen.
 7. The method ofclaim 1, further comprising the step of expressing the selected VH/VLpair in a cell.
 8. The method of claim 1, wherein the library isgenerated using at least one oligonucleotide having a sequence selectedfrom the group consisting of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 70, 71, 72, 73, 74, 75, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 101, and
 102. 9. The method of claim 1, wherein thelibrary of VH/VL pairs is a nucleic acid display library.
 10. The methodof claim 1, further comprising the step of expressing an antibodycomprising the VH/VL pair in a cell.